Adenovirus vectors have become important tools in gene therapy and for the in vivo and ex vivo cell-targeted transfer of heterologous, therapeutic genes to diseased cells or tissues, including the treatment of genetic diseases and cancer. Several properties make adenovirus advantageous gene therapy vectors. They can be produced in high titer stocks. Adenovirus can infect resting and nondividing cells, such as dendritic cells and neurons. The adenoviral genome, which is a linear, double-stranded DNA, can be manipulated to accommodate foreign genes that range in size, including reasonably large DNA inserts. They can be re-targeted to a variety of cells. They do not require host cell proliferation to express adenoviral or transgene-encoded proteins. There are no known associations of human malignancies with adenoviral infections despite common human infection with adenoviruses. As adenoviral vectors do not insert into the chromosome of a cell the effect is impermanent and less likely to interfere with the cell's normal function. Live adenovirus has been safely used for many years for human vaccines. Human adenoviruses have been used in humans as in vivo gene delivery vehicles.
However, available adenoviruses can also present serious problems when used in vivo. One drawback to adenovirus-mediated gene therapy is that decreases in gene expression are typically observed after about two weeks following administration of the vector. This loss of expression may require re-administration of the viral vector. If the same adenovirus serotype is re-administered, the host may generate neutralizing antibodies against the fiber or hexon proteins of the viral vector. Such a serotype specific anti-adenovirus response may prevent effective re-administration of the viral vector.
If viral replication is not desired, as with most gene therapy treatments, use of replication competent human adenoviruses is also problematic. For example, infection both in vivo and in vitro with the adenoviral vector can result in cytotoxicity to target cells due to the accumulation of penton protein, which is toxic to mammalian cells. Thus, in gene therapy, replication incompetent or replication defective (transgene-containing) adenovirus genomes are preferred over replication competent forms.
One approach to make a replication defective adenovirus is to inhibit viral DNA replication by disabling at least one necessary viral gene. Many currently available gene therapy adenovirus vectors are inactivated by deletion of the viral early gene region 1, (or “E1 gene”), E2A gene, E2B gene, or E4 gene. Complementation of the disabled gene by a second source will allow replication of the disabled virus. A commonly used method of complementation involves introducing the disabled (transgene-containing) adenoviral genomes into an adenoviral replication competent host cell that stably expresses the missing or mutated viral gene (e.g., the E1 gene). However, this approach has a significant risk in that the defect in the disabled genome can be replaced by homologous recombination with a wild type sequence to produce a replication competent variant.
To address problems created by homologous recombination with wild type sequences adenoviruses have been disabled by deleting most, if not virtually all, viral genes. Adenoviral vectors with only inverted terminal repeats flanking the genome (for DNA replication), an adenoviral packaging signal (to effect insertion of the completed viral genome into a completed viral capsid) and a heterologous transgene have been constructed. In this scheme, to replace the deleted viral genomic sequences a “helper” virus with the necessary complementary genes is expressed (co-transfected or co-infected) with the disabled virus in a host cell. However, this model of adenovirus disabling and complementation is inherently flawed because significant amounts of the “helper” (replication competent) adenovirus can be inadvertently encapsidated. Attempts to decrease the amount of helper partial deletion of the packaging signal in the helper virus. However, helper virus outgrowth still is a problem with these schemes.
Rapid advances in gene therapy have created a great demand for safe and effective adenoviral gene transfer vectors, particularly replication defective constructs. However, current methods for making replication defective adenoviral gene therapy vectors do not adequately address the problem of “helper outgrowth” contamination by replication competent virions. The present invention addresses these and other needs.